HHS Public Health Emergency Medical Countermeasures Enterprise (PHEMCE) indicates the development and acquisition of medical countermeasures for filoviruses as a high priority. However, currently no vaccine candidates against Ebola virus (EBOV) or Marburg virus (MARV) are nearing licensure and the need to develop a safe and efficacious vaccine against filoviruses continues. Whereas several preclinical vaccine candidates against EBOV or MARV exist, their further development is a major challenge based on i) safety concerns, ii) pre-existing vector immunity, and iii) issues such as manufacturing, dosage, and marketability. Here we propose to further develop a new and promising vaccine platform based on chemically inactivated (killed) rabies virus (RABV) virions containing EBOV glycoprotein (GP) in their envelope. Our previous research showed that immunization with such recombinant RABV virions provided excellent protection in mice against lethal challenge with the mouse adapted MA-EBOV and RABV. Moreover, the novel antigen display vehicles are highly immunogenic in non-human primates (NHP). Based on our preliminary results, the goal of this application is to develop, characterize, and extend this nove and promising filovirus vaccine platform. We propose the development and characterization a trivalent filovirus vaccine based on the killed rabies virus virions technology for use in humans t confer protection from all medically relevant filoviruses and RABV. Specifically, two additional vectors containing EBOV Sudan GP or MARV GP will be constructed in addition to the previously developed EBOV Zaire GP containing vaccine. The efficiency of these vaccines against challenge with EBOV, MARV and RABV will be studied in mice, followed by the further testing of EBOV or MARV challenge experiments in NHP. Lastly, we propose to study the required immunogenicity induced by the vaccine vectors for protection from filovirus challenge. The work proposed above toward the further development of our vaccine is performed in parallel with the transfer of the vaccine technology from a laboratory setting to GMP conditions. For this approach, we will adapt the RABV recovery system to Vero cells in a GMP facility, establish a Vero cell bank suitable for production of the vaccine as well as viral seed stocks for the three viral vectors. A production plan for the vaccine has been established, and the final goal of this translational research application is characterization of a trivalent filovirus vaccin and the production of the vaccine in sufficient quantities for a phase I clinical trial. Of note, te further characterization of this vaccine platform will establish the necessary parameters not only for a filovirus vaccine but also the use of this platform for other emerging and reemerging infectious diseases.